In recent years there has been significant progress in deciphering the molecular changes accompanying malignant transformation. A large number of tumors have been characterized as carrying a variety of chromosomal and submicroscopic genomic modifications, including activation of dominant-acting proto-oncogenes, inactivation of tumor suppressor genes and inactivation of metastasis suppressor genes. It is presumed that such tumors are descended from cell lineages that have accumulated a set of critical genetic lesions through random and relatively rare mutations at defined chromosomal locations. As an alternative tumor mechanism, some have postulated that a generalized, disseminated genomic instability, with attendant increased frequency of mutations at numerous unrelated loci, may represent a primary genetic mechanism in some tumors.
In a normal cell, multiple metabolic pathways control the overall accuracy of various functions, including DNA replication and repair, cell division and differentiation. A generalized genomic instability potentially could arise from disruption of one or more of these functions, including DNA replication, post-replicative proofreading, DNA repair, cell cycle checkpoint proteins, and DNA recombination. Mutations in genes that cause a generalized increase in the frequency of substitutions, insertions, deletions or other structural changes throughout the genome can be classified as "mutator" mutations.
Several clinical correlates have been cited in support of the theory that mutations in "mutator genes" may alter the regulation of a wide spectrum of genes, including those genes responsible for tumorigenesis. For example, individuals with the inherited disorder xeroderma pigmentosum are defective in excision repair of DNA. A clinical correlate of this condition is a predisposition of these individuals to skin cancer following exposure to ultraviolet light.
In summary, malignant transformation may involve either of the following genetic pathways:
(1) accumulation in selected cell lineages of random but relatively infrequent mutations in proto-oncogenes, tumor suppressor genes and other genes directly related to tumorigenesis. PA1 (2) mutation(s) in a mutator gene with resultant genomic instability.
There may be functional overlap in these two mechanisms in the sense that mechanism (2) may lead secondarily to mutations in known proto-oncogenes and tumor suppressor genes. However, the generalized and disseminated genomic instability of mechanism (2) may influence carcinogenesis through a wide array of known and unknown genetic mechanisms. As such, there may be little or no correlation between elevated levels of genomic instability and known mutational changes in characterized tumor-related genes. Moreover, it is conceivable that patients having tumors deriving from mechanism (2) may enjoy a relatively favorable prognosis compared to patients having tumors deriving from mechanism (1). This is due to the tendency of tumor cells having a generalized and disseminated genomic instability to continue to accumulate mutational changes, leading to cell disfunction and/or death. That is, these tumors may be relatively self-limiting in comparison to tumors deriving from mechanism (1).
While a variety of nucleic add probe-based assays are available to detect structural alterations in known tumor-related genes, there has been no reliable indicator of mechanisms involving generalized genomic instability in tumor formation. Nor has there been a reliable test to distinguish tumors arising from random mutations in tumor-related genes from those tumors arising from a more generalized genomic instability. The present inventors have discovered that monitoring the structural integrity, or sequence organization, of one or more members of selected interspersed repetitive sequence families provides a method for assessing genomic instability in tumors and for correlating such instability with tumor location and clinical prognosis.
One form of interspersed repetitive sequence in humans is the "microsatellite" class of sequences. MicroSatellite sequences of the form (CA).sub.n .cndot.(GT).sub.n constitute one of the most abundant classes of repetitive DNA families in human DNA. Approximately 50,000-100,000 (CA).sub.n -repeats are scattered throughout the human genome. Many, if not most, of these (CA).sub.n -repeats exhibit length polymorphisms. Dracopoli et al., Mapping the Human Amylase Gene Cluster on the Proximal Short Arm of Chromosome 1 Using a Highly Informative (CA).sub.n Repeat, Genomics 7:97-102 (1990); Nurnberg et al., DNA Fingerprinting With the Oligonucleotide Probe (CAC).sub.5 /(GTG).sub.5 : Somatic Stability and Germline Mutations, Hum. Genet. 84:75-78 (1989); Tautz, Hypervariability of Simple Sequences as a General Source for Polymorphic DNA Markers, Nucleic Acids Res. 17:6463-71 (1989). Although the rate of new mutations at these sites is slightly increased compared to other genomic sites, the overall rate is still quite low. In general, alleles at these sites are stably inherited from one generation to another. In fact, these markers have now been identified as one of the most useful classes of DNA polymorphism for the purpose of linkage analysis. Weissenbach et al., A Second-Generation Linkage Map of the Human Genome, Nature 359:794-801 (1992).
Although specific forms of genomic instability previously have not been correlated with specific tumors or with tumor locations and tumor prognoses, probes directed toward "minisatellite" interspersed repetitive sequence families have detected novel fragments in various malignancies. In comparison to microsatellites, minisatellites are characterized by more complex short repetitive stretches of noncoding sequences. Moreover, minisatellite loci tend to be clustered on the tips of chromosome arms. Weissenbach et al., at 794.
Southern blot analysis of genomic DNA with probes consisting of minisatellite sequences reveals an array of fragments that wiry in molecular weight. Each fragment represents multiple non-contiguous loci within the human genome. As such, these probes have been useful for multilocus fingerprinting of DNA. Jeffreys et al., Individual-Specific Fingerprints of Human DNA, Nature 316:76 (1985); Jeffreys et al., Minisatellite Repeat Coding as a Digital Approach to DNA Typing, Nature 354:204 (1991). In a similar fashion, alterations in multilocus microsatellite sequences of the form (CA).sub.n may be detected in genomic Southern blots using probes comprising various (CA).sub.n nucleotide stretches. However, a more rigorous investigation of mutational changes in individual microsatellite loci may be obtained by amplification of individual microsatellites using primer pairs directed to unique sequence flanking such microsatellites.
Additional bands or deleted bands have been observed in genomic blots of tumor DNA probed with (CAC).sub.5 and (GTG).sub.5 oligonucleotide probes. Lagoda et al., Increased Detectability of Somatic Changes in DNA from Human Tumours After Probing with "Synthetic" and "Genome-Derived" Hypervariable Multilocus Probes, Hum. Genet. 84:35-40 (1989). However, such changes were not correlated with genomic instability as related to particular tumor types, with particular anatomical groupings of tumors, or with clinical prognosis.
The sequences detected by minisatellite and microsatellite probes are not known to have any specific function. Thus, the abnormalities detected are not expected, in themselves, to be causally involved in tumor initiation or progression. However, heritable, unstable DNA elements recently have been identified as the basis of disease for three separate inherited disorders: (1) Fragile X syndrome, Kremer et al., Mapping of DNA Instability at the Fragile X to a Trinucleotide Repeat Sequence p(CCG).sub.n, Science 252:1711-14 (1991); Fu et al., Variation of the CGG Repeat at the Fragile X Site Results in Genetic Instability: Resolution of the Sherman Paradox, Cell 67:1047-58 (1991); Hirst et al., Genotype Prediction in the Fragile X Syndrome. J. Med. Genet. 28:824-29 (1991); (2) Kennedy's disease, La Spada et al., Androgen Receptor Gene Mutations in X-Linked Spinal and Bulbar Muscular Atrophy, Nature 352:77-79 (1991); and (3) Myotonic Dystrophy, Mahadevan et al., Myotonic Dystrophy Mutation: An Unstable CTG Repeat in the 3' Untranslated Region of the Gene, Science 255:1253-55 (1992); Brook et al., Molecular Basis of Myotonic Dystrophy: Extension of a Trinucleotide (CTG) Repeat at the 3' End of a Transcript Encoding a Protein Kinase Family Member, Cell 68:799-808 (1992). All available evidence suggests that amplification of a tri-nucleotide repeat is involved in the molecular pathology in each of these disorders. Although these trinucleotide repeats appear to be in non-coding DNA, they clearly are involved with perturbations of genomic regions that ultimately affect gene expression. Perturbations of various di- and tri-nucleotide repeats resulting from somatic mutation in tumor cells could also affect gene expression and/or gene regulation.
To investigate the role of disseminated genomic instability as a mechanism in tumorigenesis, it is useful to examine model systems in which tumors of potentially distinctive genetic backgrounds may be identified. Preliminary studies have indicated that different genetic mechanisms of tumorigenesis may be operative in different anatomical regions of the colorectal tract. On the one hand, evidence indicates that the process of tumorigenesis in at least some colorectal cancers proceeds through a series of genetic alterations in defined loci including both dominant and recessive acting proto-oncogenes. On the other hand, some colorectal cancers do not display such defined structural changes in known tumor-associated genes. Colorectal cancer therefore represents a useful model for analysis of disseminated genomic instability as a potential mechanism in cancer.
With respect to dominant acting proto-oncogenes in colorectal cancer, both carcinomas and the larger villous type of adenomas have shown point mutation in the ras proto-oncogene in roughly 50% of the cases. Vogelstein et al., Genetic Alterations During Colorectal-Tumor Development, N. Engl. J. Med. 319:525-32 (1988). C-myc mRNA has been found to be expressed at significantly higher levels in tumors compared to normal mucosa. Sikora et al., C-myc Oncogene Expression in Colorectal Cancer, Cancer 59:1289-95 (1987); Erisman et al., The C-myc Protein is Constitutively Expressed at Elevated Levels in Colorectal Carcinoma Cell Lines, Oncogene 2:367-78 (1988).
With respect to recessive acting proto-oncogenes (e.g., tumor-suppressor genes), several studies have demonstrated allelic loss in colorectal carcinoma. Loss of allelic heterozygosity (LOH) on a particular chromosome in cells of a tumor provides indirect evidence for the presence of a tumor suppressor gene(s) on that portion of the chromosome involved in the LOH. It has been proposed that tumor suppressor gene function can be lost through mutational inactivation of one member of an allelic pair and an accompanying chromosomal deletion that leads to physical loss or inactivation of the other member of the allelic pair. The chromosomal deletion is detected as an LOH using a polymorphic marker present in the same chromosomal region as the tumor suppressor gene. The most frequent sites of allelic loss in colon cancer appear to be on chromosomes 17 and 18 (each exhibiting LOH in nearly 75% of colorectal carcinomas); another one-third to one-half of such tumors exhibit LOH on chromosome 5. Candidate tumor suppressor genes are MCC and APC (familial adenomatous polyposis locus) on chromosome 5q, p53 on chromosome 17p and DCC (deleted in colorectal carcinoma) on chromosome 18q. Kinzler et al., Identification of a Gene Located at Chromosome 5q21 That Is Mutated in Colorectal Cancer, Science 251:1366-70 (1991); Kinzler et al., Identification of FAP Locus Genes from Chromosome 5q21, Science 253:661-64 (1991); Baker et al., Chromosome 17 Deletions and p53 Gene Mutations in Colorectal Carcinomas, Science 244:217-21 (1989), Fearon et al., Identification of a Chromosome 18q Gene That Is Altered in Colorectal Cancers, Science 247:49-56 (1990). In addition to chromosomes 5, 17 and 18, other chromosomes including chromosomes 1, 6, 8, 14 and 22 have been implicated in the genesis of colorectal cancer.
To date, results of LOH studies in colorectal cancer have shown a significant correlation with the site of the tumor. Delattre et al., Multiple Genetic Alterations in Distal and Proximal Colorectal Cancer, The Lancet 2:353-56 (1989); Kern et al., Allelic Loss in Colorectal Carcinoma, J. Am. Med. Assoc. 261:3099-3103 (1989); Offerhaus et al., The Relationship of DNA Aneuploidy to Molecular Genetic Alterations in Colorectal Carcinoma, Gastroenterology 102:1612-19 (1992). Specifically, it appears that allelic loss on chromosomes 5, 17 and 18 occurs more frequently in distal tumors than in proximal tumors. Such differences suggest that proximal and distal tumors may arise through different pathogenetic mechanisms, and that at least two genetic categories of colorectal cancer may exist. In fact, a growing body of evidence suggests that tumors of the proximal colon differ from tumors of the distal colon. Bufill et al., Colorectal Cancer: Evidence for Distinct Genetic Categories Based on Proximal or Distal Tumor Location, Ann. Int. Med. 113:779-88 (1990). This evidence, which stems from developmental and biological differences within the normal colon and various characteristics noted in colorectal cancer, includes the following:
A. Developmental.
The area extending from the cecum to the proximal two-thirds of the transverse colon is derived from the embryonic mid gut while the distal third of the transverse colon to the rectum is derived from the embryonic hind gut. Langman, J., Medical Embryology, 4th Ed. (1981). The distinct embryologic origin of these two regions is also reflected in different vascular supplies. Subsequent development leads to a number of properties that differ between the proximal and distal colon, such as the distribution of hormone producing cells.
B. Biological.
Biological differences between proximal and distal colon include differences in ability to metabolize various carcinogens, differences in the expression of cell surface antigens, and differences in enzymatic activities. Stralka et al., Cytochrome P-450 Activity and Distribution in the Human Colon Mucosa, Cancer 64:2111-16 (1980); Hughes et al., Antigen Expression in Normal and Neoplastic Colonic Mucosa: 3 Tissue-Specific Antigens Using Monoclonal Antibodies to Isolated Colonic Glands, Cancer Res. 46:2164-71 (1986); Tari et al., The Relation Between Ornithine Decarboxylase Activity and the Location of Colorectal Cancers, Gastroenterology 98:A313 (1990).
C. Epidemiological.
The relative incidence of proximal and distal tumors appears to vary in relation to the total incidence in a given region. In regions with a low incidence, proximal tumors predominate. In high risk areas, distal tumors predominate. Furthermore, the incidence of proximal tumors in the population at large appears to be increasing. Ghahremani et al., Colorectal Carcinomas: Diagnostics Implications of their Changing Frequency and Anatomic Distribution, World J. Surg. 13:321-25 (1989); Fleshner et al., Age and Sex Distribution of Patients with Colorectal Cancer, Dis. Rectum. 32:107-11 (1989).
D. Inheritance.
Colon cancer that appears in patients with familial adenomatous polyposis tends to be left-sided while those that occur in hereditary non-polyposis rectal cancer tend to be right-sided. Lynch et al., Hereditary Non-Polyposis Rectal Cancer (Lynch Syndromes I and II). II. Biomarker Studies, Cancer 56:939-51 (1985).
E. Other Proto-oncogenes.
Several studies have shown that tumors expressing deregulated c-myc are formed more frequently in the distal colon. Rothberg et al., Evidence that C-myc Expression Defines Two Genetically Distinct Forms of Colorectal Adenocarcinoma, Br. J. Cancer 52:629-32 (1985); Sikora et al., C-myc Oncogene Expression in Colorectal Cancer, Cancer 59:1289-95 (1987); Erisman et al., The C-myc Protein is Constitutively Expressed at Elevated Levels in Colorectal Carcinoma Cell Lines, Oncogene 2:367-78 (1988). On the other hand, mutations in the ras proto-oncogene occur moire frequently in proximal tumors. Offerhaus et al., The Relationship of DNA Aneuploidy to Molecular Genetic Alternations in Colorectal Carcinoma, Gastroenterology 102:1612-19 (1992).
F. Tumor DNA Content.
Analysis of DNA content by flow cytometry has shown that aneuploidy is more frequently encountered
in distal tumors than in proximal tumors. Delattre et al., Multiple Genetic Alternations in Distal and Proximal Colorectal Cancer, Lancet 2:353-56 (1989).
All of the above-referenced differences suggest the tendency of distinct pathogenetic mechanisms to be responsible for cancers of the proximal and distal colon. Although tumor suppressor genes appear to play a more prominent role in the development of tumors in the distal as opposed to the proximal colon, the underlying genetic lesions in proximal tumors have been poorly understood. Nevertheless, knowledge of the molecular genetic mechanisms involved in cancer will have an enormous impact on clinical practice and on the design of future prospective research studies covering new methods of surgical adjuvant therapy. There is an ongoing need for new prognostic indicators to more precisely identify those patients that may benefit from adjuvant therapy and those patients at high risk for tumor recurrence.